The kinetochore is a specialized protein complex that associates with centromeric DNA to promote spindle microtubule attachment to the sister chromatids, allowing for proper chromosome segregation during mitosis and meiosis. Defects in kinetochore formation can lead to missegregation of the genetic material, the basis for several types of diseases, such as cancer. Kinetochore formation is initiated through the formation of a specialized centromeric chromatin structure, which features a centromere-specific histone-H3 variant (cenH3). The mechanisms involved in targeting/maintaining centromere-specific localization of cenH3 are unknown. Using budding yeast as a model system, this proposal will focus on identifying the consequence of ubiquitin-mediated degradation on the localization and function of Cse4p, the budding yeast cenH3. This will involve characterizing the phenotypes associated with a Cse4p mutant that is incapable of being degraded as well as determining whether the kinetochore protects centromere-associated Cse4p from ubiquitin-mediated proteolysis. A second objective of this proposal is to further investigate the role of Cse4p in chromosome segregation using conditional mutant alleles of cse4.